This project will use voltage clamp experiments to study the ionic currents that generate the action potential of cardiac Purkinje fibers. In particular, the 4-aminopyridine (4-AP) and the chloride sensitive components of membrane current will be characterized and possible membrane control mechanisms will be considered. Shortened calf and sheep Purkinje fibers will be voltage clamped using the two-and three-microelectrode voltage clamp methods. The three microelectrode voltage clamp will be used to test the adequacy of the longitudinal space clamp in these preparations (Kass, et. al., J. Physiol. 290:201 1979). Measurements of the twitch tension will be used to assay the intracellular calcium ion activity. 4-AP will be used to dissect out a component of the net membrane current. The 4-AP sensitive current will be identified by subtracting the current remaining in the presence of 4-AP from control currents for matching voltage clamp steps. Single step and pre-step protocols will be used to study the time- and voltage-dependence of the inactivation of this current component. The time- and voltage-dependence of the twitch tension will be compared with that of the 4-AP sensitive current to test for a role of intracellular calcium in the gating of this current. The 4-AP sensitive component of membrane current noise will be studied by fluctuation analysis to measure the mean open channel lifetime and the single channel conductance. Replacement of the extracellular chloride with either methylsulfate or methanesulfonate will be done to study the chloride sensitive component. The kinetics of this current will be studied in experiments similar to those proposed with 4-AP. The possibility that the chloride sensitivity of this current arises from changes in the intracellular pH (pHi) caused by the chloride reduction, and not by the chloride replacement per se, will be tested by measuring the pHi with a pH sensitive microelectrode during voltage clamp experiments. In these experiments the effects of independently changing the extracellular chloride or the pHi will be examined.